Wogonin Protects Against Cisplatin-induced Acute Kidney Injury By Targeting RIPK1-mediated Necroptosis

Acute kidney injury(AKI)is mainly characterized by an increased inflammatory response and the destruction of innate cells in the kidney,which in turn cause a drastic derangement of renal function.Normal renal tubular epithelial cells(HK2)are in good shape and arranged in neat rows,while injured cells are in irregular shape,fall off and the nucleus are shrinkage.Pathological features include recruitment of macrophages,release of inflammatory cytokines,and delay of death and proliferation of kidney-resident cells.Ischemia and reperfusion(I/R),nephrotoxic drugs,sepsis and other causes can cause AKI,however,the specific mechanism of AKI and effective treatment need further exploration.Renal cells can undergo apoptosis,necrosis,or autophagy in AKI.Recent studies have reported that necroptosis,not apoptosis,plays an important role in AKI and triggers a serious inflammatory response.Cells which undergo necroptosis not only have necrotic structural features,but also are regulated by death signaling pathways.RIPK1/RIPK3/MLKL axis is currently widely recognized as major regulators of necroptosis.To date,effective treatment for AKI is lacking.In view of the significant pharmacological effects and relatively low toxicity of traditional Chinese medicine(TCM),it has been used to treat various diseases.The research team screened more than ten traditional Chinese medicines for renoprotective effects against cisplatin-induced kidney injury.Preexperiment results showed that wogonin had the best protective effect against cisplatininduced AKI.It has been reported that wogonin has anti-inflammatory,anti-oxidation and anti-tumor effects.In order to study the therapeutic effect and protective mechanism of wogonin on AKI,the experiment is divided into the following parts.1.The protective effect of wogonin on acute kidney injury in mice.Male C57BL/6 mice(6-8 weeks)were randomly divided into normal group,model group,low dose group,middle dose group and high dose group,and every group was 8 mice.Mice in model group and therapeutic groups were given acute 20 mg/kg cisplatin by intraperitoneal injection.At the same time,mice in therapeutic groups were treated by wogonin at 12.5,25 and 50 mg/kg by intraperitoneal injection daily,respectively,normal saline as a control.All the mice were sacrificed three days later under anesthesia.The kidneys and blood were collected for further analysis.In order to observe the protective effect of wogonin on AKI,pathological examination of renal tissue was performed by PAS staining.Serum levels of Cre and BUN were measured by Cre and BUN kit.The expression of KIM-1 in kidney was detected by RT-PCR.The number of F4/80+ cells was detected by immunohistochemistry and the expression of inflammatory cytokines was measured by RT-PCR.PAS staining and scoring showed that compared with the model group,treatment of wogonin by 12.5,25 and 50 mg/kg daily could obviously reduce the tubular necrosis and dilation.The Cre and BUN were decreased obviously in the treatment group mice.Meanwhile,wogonin significantly reduced the protein and m RNA levels of KIM-1 induced by cisplatin.The results of F4/80+ staining showed that wogonin can reduce the infiltration of macrophages.The results of western blot suggested that wogonin can inhibit the activation of NF-κB pathway and the expression of TNF-α stimulated by cisplatin.Cisplatin-induced inflammatory factors including IL-1β and IL-6 are also supressed by wogonin.Electron microscopy further confirmed that wogonin can significantly improve cisplatin-induced disruption of nuclear structure and intracellular organelle abnormalities.The above results suggested that wogonin has a certain therapeutic effect on AKI and inhibit inflammatory responses.2.The protective effect of wogonin against cisplatin-induced HK2 cell injury in vitro.MTT assay was used to detect the effects of different concentrations of wogonin(0,0.625,1.25,2.5,5,10,20,40μg/ml)on the survival rates of HK2 cells.The results showed that wogonin has no cytotoxicity when the concentration was lower than 2.5μg/ml.Cells were treated with cisplatin and incubated with wogonin simultaneously.MTT assay and LDH assay showed that wogonin could protect against the inhibitory effect of cisplatin on the viability of HK2 cells by a dose-dependent manner.The next experiments will use the 2.5 μg/ml.We know that cisplatin works as clinically common antineoplastic drug.It is worth mentioning that the results of MTT assay showed that wogonin not only does not affect the anti-tumor effect of cisplatin,but even enhance the effect of cisplatin on Hep G2.Therefore,wogonin can be effectively used as an adjuvant for renal protection in AKI in the treatment of cancer patients with cisplatin.Acute kidney injury model in vitro was established by cisplatin-induced HK2 cells.Electron microscopy images showed that wogonin could improve cisplatin-induced nuclear swelling and organelle loss.Flow cytometry and PI/ Annexin V immunofluorescence showed that cisplatin could induce apoptosis and necrosis in HK2 cells.Wogonin could reduce the number of cells which undergo early apoptosis,late apoptosis and necrosis.The expression of KIM-1 was detected by western blot and immunofluorescence.The results showed that wogonin can effectively reduce the expression of KIM-1 stimulated by cisplatin.The protein level of TNF-α was detected by immunofluorescence,and the m RNA levels of inflammatory cytokines such as TNF-α and IL-1β were detected by RT-PCR.The results showed that wogonin largely inhibited the release of inflammatory cytokines.The ELISA assay for detecting IL-6 and IL-8 further demonstrated the protective effect of wogonin against AKI.3.The effects of wogonin on key proteins of necroptosis during AKI and explore the relationship between wogonin and RIPK1 by molecular docking.Western blot was used to detect the expression of RIPK1,RIPK3 and p-MLKL in different groups.The results showed that wogonin can effectively inhibit the expression of RIPK1,RIPK3 and p-MLKL induced by cisplatin in vitro and in vivo.The molecular docking suggested that wogonin may be an ideal inhibitor of RIPK1.Because it can occupy the ATP binding pocket of RIPK1 enzyme and thus inhibits the activity of RIPK1,which indicates that wogonin may target RIPK1-regulated necroptosis and improves cisplatininduced AKI.In view of apoptosis of cells in AKI,we detect the index of apoptosis in order to gain a more complete understanding of the protective mechanism of wogonin.The results showed that wogonin can only mildly inhibit cisplatin-induced cleavedcaspase3/8 expression,while the necroptosis proteins were significantly inhibited.4.Further explore the protective mechanism of wogonin against acute kidney injury by interfering with RIPK1 and RIPK3.To further demonstrate the inhibitory effect of wogonin on RIPK1/RIPK3 in cisplatininduced AKI,RIPK1 and RIPK3 were separately blocked to explore whether protective effect of wogonin was affected.The results showed that Nec-1,RIPK1 inhibitor,further reduced the expression of RIPK1/RIPK3/p-MLKL and inhibited the expression of KIM-1,TNF-α and IL-6 induced by cisplatin.However,when RIPK1 was blocked,wogonin could not further down-regulate the expression of KIM-1 and IL-6,which suggests that wogonin may function through RIPK1.In addition,the expression of RIPK3 was silenced by transfection with RIPK3 sh RNA.The results showed that RIPK3 silencing could reduce the protein levels of RIPK3 and p-MLKL without affecting the level of RIPK1.At the same time,RIPK3 silencing could significantly reduce the expression of KIM-1,TNF-α and IL-6.And wogonin can not further play a protective effect.The above experimental results further verify the protection mechanism of wogonin.Taken together,this study has shown that wogonin protects against cisplatin-induced AKI through inhibiting RIPK1-mediated necroptosis.Moreover,wogonin does not affect or even promote the anti-tumor effect of cisplatin.Therefore,wogonin can effectively serve as an adjuvant for renal protection in the treatment of cancer patients with cisplatin.